Asp-52 in combination with Asp-398 plays a critical role in ATP hydrolysis of chaperonin GroEL

Abstract

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼ 150 h (∼ 6 days), providing a good model to characterize the football-shaped complex.

Keywords: ATPase; Chaperone; Chaperonin; GroEL; GroES; Protein Aggregation; Protein Folding; Protein Misfolding.

FIGURE 1.

Comparison of nucleotide binding site of a GroEL subunit and a thermosome α subunit. A, the structure around the nucleotide binding site of the GroEL subunit in the cis-ring from GroEL-GroES-ADP-AlF_x complex (PDB code 1SVT (29)). Equatorial, intermediate, and apical domains are colored blue, green, and pink, respectively. B, the structure around the nucleotide binding site of the thermosome α subunit structure from thermosome-ADP·AlF₃ complex (PDB code 1A6E (7)). Side chains of Asp-52/Asp-398 in A and Asp-63/Asp-390 in B are drawn as stick models in orange. ADP and AlF_x are colored red and yellow as stick models, and the Mg²⁺ is green spheres.

FIGURE 2.

ATPase activity of Asp mutants of GroEL. ATPase activity of GroEL^WT, GroEL⁵², GroEL³⁹⁸, and the double mutant GroEL^52/398. Aliquots of 0.2 μm GroEL and 0.6 μm GroES were used for the ATPase activity assay at 25 °C. rLA, reduced lactalbumin as a model substrate protein.

FIGURE 3.

GroEL-assisted folding of rhodanese and MDH. A, denatured MDH or rhodanese were diluted in buffer containing GroEL and GroES. The recovery of activity was measured at 60 min after the initiation of folding by the addition of ATP. Activity is expressed as the fraction of the final yield of the MDH or the rhodanese recovery by GroEL^WT at 60 min. B, time course of recovery of rhodanese activities. Denatured rhodanese was diluted in buffer containing ATP in the presence of GroEL variants (GroEL^WT, solid circles; GroEL⁵², open circles; GroEL³⁹⁸, open squares; GroEL^52/398, solid triangles) and GroES. At time 0, ATP was added to the mixtures to initiate folding. An aliquot was withdrawn and assayed for rhodanese activity at the indicated times. Activity is expressed as the fraction of the final yield of the rhodanese recovery by GroEL^WT at 60 min.

FIGURE 4.

Encapsulation of rhodanese within cavities of the GroEL^52/398-GroES complex. Mixtures of GroEL mutants saturated with heat-denatured rhodanese, GroES, 200 mm glucose, and ATP were incubated for 3 s (lanes 1 and 4), 1 min (lanes 2 and 5), or 120 min (lanes 3 and 6). In the experiment shown in lanes 1 and 4, hexokinase was added to the aliquot after 3 s, and the reaction was left for another 60 min. The aliquots were subjected to ultrafiltration (100-kDa cutoff) and SDS-PAGE. Gels were stained with Coomassie Brilliant Blue. The GroES/GroEL molar ratios determined by the band intensities were 1.7 (lane 1), 1.9 (lane 2), 1.2 (lane 3), 1.7 (lanes 4 and 5), and 2.2 (lane 6).

FIGURE 5.

Electron micrographs of the negatively stained GroEL^52/398-GroES complex in the presence of ATP. Arrowheads show the symmetrical football complexes. The scale bar represents 60 nm.

FIGURE 6.

Stability of symmetric GroEL^52/398-GroES football complex. A, decay of the symmetric GroEL^52/398-GroES football complex. The GroEL^52/398 ATP football complex was generated by mixing GroEL^52/398 with unlabeled GroES at a 1:2 molar ratio in the presence of ATP (t = 0 min). After an incubation for 5 min, the mixture was applied to a gel filtration column to isolate the ATP football complex. The isolated ATP football complex (0.3 μm) was mixed with Cy3-labeled GroES (GroES^Cy3, 0.6 μm) and ATP (1 mm) at the indicated times and then separated by gel filtration HPLC with fluorescence detection (20, 22). The trace labeled control represents a direct mixing of unliganded GroEL^52/398 (0.3 μm) with 0.6 μm GroES^Cy3 and 1 mm ATP. a.u., absorbance units. B, time course of ATP hydrolysis by the isolated GroEL^52/398 ATP football complex and quantification of the GroES bound to the GroEL^52/398 ATP football complexes. The ratio of ATP or ADP to the total nucleotide is shown. The integrated peak areas in the traces obtained by the binding assay of GroES^Cy3 were calculated. The amounts of GroES bound to unliganded GroEL^52/398 in the presence of ATP was taken as 200%.